The invention relates to the use of deglycosylated detectors, especially secondary antibodies, for the determination of sugar structures of proteins, especially recombinant proteins. It further relates to the use of deglycosylated enzymes in the determination of sugar structures with the aid of an enzyme-substrate reaction; the invention further relates to a method of determining the sugar structures of proteins, a sugar determination kit and the use of said kit for the determination of sugar structures, especially of recombinant therapeutic proteins, preferably immunoglobulins.
Recombinant therapeutic proteins are well-known in the prior art. For example, human proteins such as erythropoietin or antibodies have been recombinantly produced in CHO cells in the prior art. While this technology ensures reproduction of the protein amino acid sequence encoded in the DNA, the post-translational modifications of the recombinant protein, such as glycosylation, are variable depending on the cell clone and the culture conditions. When providing recombinant proteins, especially therapeutic proteins, it has to be kept in mind that, depending on the glycosylation, the sugar structures or protein glycosylation will give rise to a tissue-, cell-, development- and/or species-specific and typical behavior. Essential structural properties having central importance in therapeutic use are determined by the sugar structures of the proteins. For example, well-known recombinant therapeutic proteins are complete, i.e. intact, monoclonal antibodies.
To date, no method has been described in the prior art that would allow rapid and automatable determination of sugar structures, especially in recombinant therapeutic proteins such as intact monoclonal antibodies.
Up to now, complex procedures such as HPLC, MS or CE have been used in the prior art after removing the sugars. To this end it is necessary to remove the sugar structures from the protein using chemical or enzymatic hydrolysis. Although enzymatic procedures allow selective removal of specific structures, they are complex in practice and susceptible to interference. After removing the sugars, separation from the protein is necessary, which is frequently carried out using column chromatography. The steps of preparation and the sensitivity of subsequent analytic procedures, which frequently is low, result in the necessity of using more than a small amount of recombinant protein (500 to 1000 μg), which appears disadvantageous in practice. The determination of sugar structures often requires combining a number of analytic procedures, e.g. a mass-spectrometric investigation using MALDI-MS, which furnishes qualitative results and is not able to differentiate between hexoses, and a quantitative determination of defined sugars using HPLC or photometric methods. The use of the latter methods depends on the availability of pure glycans as comparative standards.
Inter alia, a method of detecting the terminal glycosylation pattern of IgG has been disclosed in the prior art. To this end, a sample comprising the protein to be investigated is immobilized on nitrocellulose. Thereafter, the proteins, preferably IgG, are treated in such a way that the sugar residues (Gal, GlcNAc) are liberated. The IgGs in the sample are detected using labeled lectins, said labeling in turn being effected by means of enzymes (WO 89/04490).
Another prior art method comprises immobilization of lectins on a plate and detection using a conjugate of secondary antibody and enzyme. In this method the lectin is selected in such a way that it can only recognize an abnormal sugar structure, for which reason it is not suitable for the determination of IgGs with galactose, fucose and other sugars (WO 98/16825).
Further, a method of detecting a glycoprotein using labeled lectins has been described in the prior art, wherein a so-called capture antibody (Fab portion) is immobilized first. The Fc portion thereof is removed so as to discard sugars that might impede the measuring operation. Disadvantageously, this method (WO 03/087821) has failed to solve the problem of Fab glycosylation and, in addition, the glycosylation of the enzyme in the determination of the sugar structures has not been accounted for.
EP 0 399 464 A2 discloses five methods wherein specific sugar residues are determined using plastic particles. Due to possible sugar structures on the substances used, these methods have failed to furnish satisfactory results as is the case with the method according to EP 0 662 611 A2 wherein glycoconjugates in a sandwich structure with lectins are investigated by initially immobilizing a capture antibody present in chemically treated form so as to inactivate the sugar and avoid false background signals.
Further prior art is identified in Lundy et al., 1999, WO 2004/040307, EP 1 460 452 and WO 03/087831 wherein various assays for the detection of glycoproteins have been described. Disadvantageously, the latter do not allow good determination of sugar structures of proteins because the individual measured signal cannot be sufficiently distinguished from the background noise.
None of the prior art methods are suitable for reliable and effective determination of sugar structures, especially in recombinant proteins such as immunoglobulins, particularly IgG.